Hepatitis C virus (HCV) is an important cause of liver disease. Current therapies are inadequate. A cocktail of multiple drugs, each targeting an independent viral function offers the best chance for effective pharmacologic control. Our long-term objectives are to better understand the molecular virology of HCV, increase the repertoire of new targets that can be translated into novel drug classes for inclusion in future anti-HCV cocktails. We have identified and genetically validated two new targets within NS4B: an arginine-rich-like motif mediated RNA binding activity that is specific for the 3' terminus of the viral negative RNA strand (3'term(-)); and an amphipathic helix, termed 4BAH2, with a dramatic ability to promote lipid vesicle aggregation, an ideal candidate biochemical activity for creating the membranous web, the HCV replication platform. We then identified small molecule inhibitors of these two targets with significant anti-HCV activity: a first generation H1 antihistamine-clemizole-potently inhibits NS4B RNA binding and exhibits dramatic in vitro synergy with the HCV NS3 protease inhibitor SCH503034. Two distinct compounds-A2 and C4-were each found to be potent specific inhibitors of 4BAH2-mediated lipid vesicle aggregation. Finally, we recently developed a new method for rapidly mapping RNA secondary structure, affording new ways to study RNA elements such as the target of NS4B binding. Our overall hypothesis is that two different newly described functional activities within NS4B are each essential for mediating NS4B's role in HCV replication. Thus approaches designed to disrupt the function of these domains may be potentially used to inhibit HCV replication in patients. More specifically, we hypothesize: 1) clemizole's antiviral activity is distinct from its antihistamine activity; 2) the mechanism of action of A2 and C4 can be further validated by analysis of resistant mutants and derivatives of these compounds; 3) while A2 and C4 both target the same 4BAH2, they do so via distinct mechanisms, where one targets 4BAH2's interaction with membranes, and one targets 4BAH2's interaction with itself; 4) NS4B RNA binding inhibitors are broadly synergistic with NS3 protease inhibitors; 5) both NS4B RNA binding inhibitors and 4BAH2 inhibitors represent attractive potential new drug classes for future anti-HCV therapeutic cocktails designed to maximize efficacy and minimize resistance; 6) 4BAH2's lipid vesicle aggregating activity reflects a biochemical activity important for membranous web formation that is amenable to both genetic and pharmacologic disruption, and the specific amino acids critical for this activity can be identified by mutational analysis; 7) the secondary structure of the HCV 3'term(-) is altered upon interaction with NS4B or clemizole, or upon mutation to escape clemizole inhibition; 8) expression of wild-type and drug-resistant forms of NS4B can enable critically important 3D structure determinations. To test these hypotheses, we propose to further study these compounds' mechanism of action, determine their potential as critical components of future anti-HCV cocktails, and better understand the structure and function of these compounds' targets within NS4B and the HCV life cycle. PUBLIC HEALTH RELEVANCE: HCV remains an important cause of worldwide liver disease for which current therapies are inadequate. We have discovered two new targets within the HCV NS4B protein and identified small molecule inhibitors of these targets. We propose to further study these compounds' mechanism of action, better understand the structure and function of these compounds' targets within NS4B and the HCV life cycle, and determine these compounds' potential as critical components of future anti-HCV cocktails.